Analytical and diagnostic determinations can be performed on liquid samples by means of optical assays based on the detection of analytes in a sample, such as e.g. nucleic acids, peptides, proteins, antibodies, hormones, or drugs. One important application of optical assays is the field of immunology, in which the analyte is detected with the aid of a specific antibody, which is capable of binding to the analyte to form optically detectable complexes, e.g. by labeling the analyte with a fluorophore, or by providing a fluorophore-labeled antibody before the optical detection. The detection of the fluorophores may be performed by means of a fluorescence reader, which is capable of illuminating the assay support substrate with an exciting light source and of detecting the fluorescent light emitted from the fluorophores.
An optical assay involving detection of fluorescent light emitted from fluorophores is performed by an optical assay arrangement comprising a sample supporting substrate and a fluorescence reader. The fluorescence reader comprises a source and detector for electromagnetic radiation within the optical wavelength region (i.e. between approximately 40 nm and 1 mm), and suitable optical filters and wave-guiding means. The sample support comprises a substrate of e.g. a polymeric material having a high optical transmittance in the wavelength ranges of the exciting light and of the emitted light, and may also have a high absorbance of other wavelengths. The substrate is provided with one or more reaction site areas, comprising spots and/or lines of probe molecules, e.g. of an antibody, providing binding sites for molecules of the analyte, i.e. the target molecules, that may be present in a sample. The substrate may further be provided with a pattern of protruding microstructures forming e.g. micro pillars or micro posts, which may be arranged to form a capillary flow path for the sample.
When the sample is brought in contact with the capture molecules on the support surface, and the fluorescent or phosphorescent antibody detection conjugate, optically detectable spots or lines will be formed. Fluorescent or phosphorescent light will be emitted when the substrate is illuminated with the exciting light source of the fluorescence reader, thereby indicating that a reaction has occurred between the target molecules of the sample and the probe molecules of the reaction sites. Fluorescence and phosphorescence may be defined as the emission of electromagnetic radiation resulting from absorbed exciting electromagnetic radiation, the fluorescent light lasting less than 1×10−8 s after the excitation, and phosphorescent light lasting longer, i.e. is decaying more slowly after the exposure to the exciting light.
In fluorescence (and phosphorescence), the exciting radiation normally has a shorter wavelength (i.e. higher energy) than the emitted radiation, although the reverse may be true for multi-photon fluorescence. The fluorescent behaviour may be studied in a steady state or time-resolved, and fluorescence spectroscopy involves e.g. single-and multi-photon fluorescence, FRET (fluorescence resonance energy transfer), and fluorescence up-conversion. In fluorescence assays, the wavelength of the exciting and the emitted radiation depends on the type of fluorophore, which may be of an organic or inorganic origin, e.g. cyanine dyes, fluorescin dyes or nanocrystals. As an example, the common fluorophore Cy-5™ (GE Healthcare) is typically excited with 649 nm, and the emitted light is measured at 670 nm. The difference in wavelength between the excitation maxima and the emittance maxima is commonly referred to as the Stokes shift.
In optical assays, the concentration of an analyte in the sample may be determined by measuring the intensity of emitted fluorescent or phosphorescent light, by means of the detector device of a fluorescence reader, thereby enabling quantitative measurements. Consequently, the efficiency of the illumination of a reaction site area with exciting light, as well as the efficiency of the collection of the emitted fluorescent light, will have an effect on the performance of the optical assay.
Further, the reaction sites on a substrate surface may be provided with an array of spots or lines of different probe molecules, binding different target molecules. Therefore, a fluorescence reader may be designed to be capable of determining the presence of several analytes in a sample, by means of different fluorophores, or by using space separation of the probe molecules.
A fluorescence reader can be arranged to perform the detection of fluorescent emitted light by scanning the reaction site area or to detect an image of the entire reaction-site area as a two-dimensional array of pixels. A scanning fluorescence reader scans the surface of the sample substrate by a relative movement between the optical means and the sample substrate, and the optical means preferably comprises a narrowband exciting light source, such as a laser, a LED or a white light source provided with spectral filters, from which the light is focused on each individual detection site. The emitted fluorescent light from each detection site is focused on an optical detector, such as e.g. a photodiode or a PMT (photomultiplier tube). An imaging fluorescence reader is capable of detecting a two-dimensional array of pixels and the optical means comprises an exciting light source for illuminating a large part of the surface area (or the entire surface area) of the sample substrate, and a detector capable of detecting emitted light from the entire detection site-area simultaneously, e.g. a CCD (Charged-Coupled Device)-imager, which utilizes MOS (Metal-On-Semiconductor)-technology.
An optical reader is described in WO 01/575501, which discloses optical imaging of an analyte containing sample on a transparent substrate. The optical reader comprises an exciting energy source to stimulate emission of detectable light from the sample, and the substrate is provided with a reflective surface located below the sample to reflect the emitted light into the detection means.
A light detecting optical device is disclosed in WO 99/465961, comprising a light conducting body coupled to a slide, thereby improving the light collecting efficacy.
WO 03/103835 describes sample substrates provided with protruding micro posts arranged to form a capillary flow path for the sample.
It is an object of this invention to provide an improved fluorescence/phosphorescence reader capable of an efficient illumination of the reaction site area of the substrate and an efficient collection and detection of the emitted light, thereby achieving a high performance optical assay arrangement.